Components and Levels Tested with RNaseAlert® Technology

The following tables contain the compounds tested and indicate the compatibility of the compound tested with RNase A activity.

Experimental procedure: 45 µl of each listed reagent was added to an RNaseAlert® tube containing lyophilized substrate plus 5 µl of 10X RNaseAlert® Reaction Buffer. These mixtures were incubated for 1 hr at 37°C and then inspected for fluorescence on a UV transilluminator. Sample fluorescence was marked "-" in the "Before Addition of RNase A" if there was no visible glow and "+" if there was visible green glow.

After UV inspection and scoring, the "-" samples were further tested by adding 100 pg of RNase A and incubating an additional hour at 37°C. These tubes were then scored as above and results listed in the "After Addition of RNase A" column. RNase A was not added to enzyme solutions possessing RNase activity.

Reagents that are compatible with the RNaseAlert® technology are the ones marked "-" in the "Before Addition of RNase A" column and "+" in the "After Addition of RNase A" column.

This is not a comprehensive list of compatible reagents. It serves to illustrate some typical data using the RNaseAlert® Technology. More sophisticated monitoring can be performed with the RNaseAlert® QC System or RNaseAlert® QC System v2 using a fluorometer. Note that tips and solid surfaces are frequently positive for RNases if gloveless hands have simply touched them.

Ambion Kit Components Tested with the RNaseAlert® Technology

Before Addition of RNase A*

After Addition of RNase A*

Comments

DNaseZap™ Solution 1

-

+

Gel Loading Buffer II

-

-

Not recommended. The dye in the loading buffer quenches RNaseAlert® fluorescence.

MAXIscript™ Transcription Buffer, 1X

-

+

MEGAscript™ Reaction Buffer, 1X

-

+

NorthernMax™ Transfer Buffer

-

+

NorthernMax™-Gly Gel Loading Buffer

-

-

Inhibits RNase A activity.

NorthernMax™-Gly Gel Running Buffer, 1X

-

+

Random Decamers, 10X

-

+

Salmon Sperm DNA (sheared)

-

+

THE RNA Storage Solution

-

+

*Reagents that are compatible with RNaseAlert® technology are the ones marked "-" in the "Before Addition of RNase A" column and "+" in the "After Addition of RNase A" column.

Chemicals and Reagents Tested with the RNaseAlert® Technology

Before Addition of RNase A*

After Addition of RNase A*

Comments

Acetone 10%

-

+

Acrylamide/Bis-acrylamide 19:1, 4%

-

+

Acrylic acid

-

-

Inhibits RNase A activity.

Agarose Gel Loading Buffer, 10%

-

+

Ammonium acetate, 500 mM

-

-

Inhibits RNase A activity.

Ammonium persulfate, 1%

-

-

Inhibits RNase A activity.

ATP, 1 mM

-

+

Betaine, 500 mM

-

+

Bromophenol Blue, 0.08%

-

-

Not recommended. Bromophenol blue quenches RNaseAlert® fluorescence.

Cesium Chloride

-

-

Inhibits RNase A activity.

Chloroform

-

-

Coommassie Destain

-

-

Inhibits RNase A activity.

DEPC-treated water

-

+

DMSO 10%

-

+

EDTA 100-500 mM

-

+

500 mM inhibits RNase A activity.

Ethanol, 10%

-

+

Ethidium bromide

-

+

Not recommended. Ethidium bromide has orange/red fluorescence that makes reading the fluorescence of the RNaseAlert® substrate difficult.

Glycerol

-

+

Glycerol, 10%

-

+

Glycine

-

+

GTP, 7.5 mM

-

+

Guanidinium hydrochloride, 100-400 mM

-

+

400 mM inhibits RNase A activity.

Guanidinium thiocyanate, 100-400 mM

-

+

200 mM inhibits 5 pg of RNase A

Magnesium acetate, 200 mM

-

-

Inhibits RNase A activity.

Magnesium acetate, 30-100 mM

-

+

Magnesium chloride, 100 mM

-

+

Mineral Oil

-

+

MOPS Gel Running Buffer, 10X

-

+

NorthernMax Buffer (10X)

-

-

Inhibits RNase A activity.

NP40, 1%

-

+

Nuclease-free water (not DEPC-treated)

-

+

PBS, 0.1X

-

+

PBS, 1X

-

+

PEG 8000, 4%

-

+

Phenol

-

-

Incubation with RNase A yielded slight violet fluoresecence.

Potassium acetate, 100 mM, pH 8.0

-

+

Potassium chloride, 200 mM

-

-

Inhibits RNase A activity.

Potassium phosphate, 100 mM

-

-

Inhibits RNase A activity.

RNAlater™

-

-

SDS, 1%

-

+

Sodium Acetate, 300 mM, pH 5.2

-

-

Inhibits RNase A activity.

Sodium Chloride, 10-200 mM

-

+

Sodium Chloride, 500 mM

-

-

Inhibits RNase A activity.

Sodium Deoxycholate, 0.4%

-

+

Sodium phosphate, 2 mM

-

+

TBE, 1X

-

+

TE, pH 8

-

+

Tris, 100 mM, pH 8.0

-

+

Tris, 100 mM, pH 7.0

-

+

Tween, 1.0%

-

+

Urea polyacrylamide gel solution, 6%

-

+

Yeast Lysis Buffer

-

+

Zinc acetate, 100 mM

-

-

Inhibits RNase A activity.

*Reagents that are compatible with the RNaseAlert® technology are the ones marked "-" in the "Before Addition of RNase A" column and "+" in the "After Addition of RNase A" column.

Materials Tested with the RNaseAlert® Technology

Before Addition of RNase A*

After Addition of RNase A*

Comments

1 week old coffee with mold

+

ND*

Glowed like the moon.

Agilent Bioanalyzer electrodes, touched with bare hands

+

ND

Positive.

Frank's bare hands **

+

ND

No RNase activity detected.

Gary's bare hands**

+

ND

Slight glow.

George's bare hands

+

ND

Glowed brightly.

LB with bacterial funk, contaminated

+

ND

Glowed like a Texas firefly in August.

Pipet tips touched with bare hands

+

ND

Positive.

Saliva

+

ND

Positive.

* ND = Not determined.
** 100 µl of RNase-free water was placed in the palm of the subject's bare hand. 5 µl of that water was used in the assay according to the kit protocol.